BMP2 genetically engineered MSCs and EPCs promote vascularized bone regeneration in rat critical-sized calvarial bone defects

Abstract

Current clinical therapies for critical-sized bone defects (CSBDs) remain far from ideal. Previous studies have demonstrated that engineering bone tissue using mesenchymal stem cells (MSCs) is feasible. However, this approach is not effective for CSBDs due to inadequate vascularization. In our previous study, we have developed an injectable and porous nano calcium sulfate/alginate (nCS/A) scaffold and demonstrated that nCS/A composition is biocompatible and has proper biodegradability for bone regeneration. Here, we hypothesized that the combination of an injectable and porous nCS/A with bone morphogenetic protein 2 (BMP2) gene-modified MSCs and endothelial progenitor cells (EPCs) could significantly enhance vascularized bone regeneration. Our results demonstrated that delivery of MSCs and EPCs with the injectable nCS/A scaffold did not affect cell viability. Moreover, co-culture of BMP2 gene-modified MSCs and EPCs dramatically increased osteoblast differentiation of MSCs and endothelial differentiation of EPCs in vitro. We further tested the multifunctional bone reconstruction system consisting of an injectable and porous nCS/A scaffold (mimicking the nano-calcium matrix of bone) and BMP2 genetically-engineered MSCs and EPCs in a rat critical-sized (8 mm) caviarial bone defect model. Our in vivo results showed that, compared to the groups of nCS/A, nCS/A+MSCs, nCS/A+MSCs+EPCs and nCS/A+BMP2 gene-modified MSCs, the combination of BMP2 gene -modified MSCs and EPCs in nCS/A dramatically increased the new bone and vascular formation. These results demonstrated that EPCs increase new vascular growth, and that BMP2 gene modification for MSCs and EPCs dramatically promotes bone regeneration. This system could ultimately enable clinicians to better reconstruct the craniofacial bone and avoid donor site morbidity for CSBDs.

Figure 1. Phenotype identification of MSCs and EPCs.

(A) Flow cytometry analysis for MSCs. The red open histograms indicated the cells which were stained positive for the undifferentiated MSCs markers -CD44 and CD90 and negative for CD 31 and CD34. The black open histograms showed isotype-matched control staining. (B) Flow cytometry analysis for EPCs. The red open histograms indicated the cells which were stained positive for the markers of undifferentiated EPCs - CD133 and CD34, and negative for CD31 and CD11b. The black open histograms showed isotope-matched control staining. (C-E) Fluorescence images of EPCs cultured for 14 days and stained with (C) Dil-Ac-LDL, (D) lectin and (E) DAPI. Bar = 50 µm. (F) Overlay of images of C, D and E.

Figure 2. Co-culture of EPCs and MSCs in nCS/A scaffold.

(A) MTS cell viability assay. The cell number was 5×10⁴ per scaffold for the four groups. Data represent the mean+SD for  = 8. p>0.05: between any two groups. (B) Optimization of the co-culture ratio of EPCs and MSCs. Cells were plated at EPCs:MSCs ratios of 0:1 (MSCs alone, indicate as M),1:0 (EPCs alone, indicate as E), 2:1 1:1, 1:2, 1:5 and 1:10 with a total cell number of 1×10⁵ and then cultured in EGM/CM (EGM media: complete media ratio of 1:1) or EGM/OS media (EGM media: OS media ration of 1:1) for 7 days for ALP activity assay. ND means no significant difference between each of those three groups (p>0.05). Significant difference from the group of 1:1 is indicated by *(p<0.05). # p<0.05: all other groups between the cells treated with EGM/CM and EGM/OS media except EPCs group, which has no difference between the cells treated with EGM/CM and EGM/OS media. (C) ALP activity. MSCs, EPCs, BMP2 gene-modified MSCs and BMP2 gene-modified EPCs were respectively plated in 24-well plates at a density of 2.5×10⁴ cells/cm² in the different combination. The cells were induced with EGM/OS media (EGM media: OS media ration of 1:1) for 7 days for ALP assay. Data represent the mean+SD of n = 6 samples. In the groups, M: MSCs, E: EPCs, B2: BMP2, M+E: MSCs+EPCs, B2/M: BMP2 gene-modified MSCs, B2/M+E: EPCs and BMP2 gene -modified MSCs. B2/(M+E): BMP2 gene-modified MSCs and BMP2 gene-modified EPCs. ND means no significant difference between any two groups of those three groups (p >0.05). Significant difference from the group of B2/(M+E) is indicated by * (p <0.01).

Figure 3. qRT-PCR analysis of osteoblast and endothelial marker genes.

MSCs, EPCs or MSCs and EPCs with or without BMP2 gene modification (1∶1) were plated in 24-well plate as a density of 2.25×10⁴ cells/cm² as described in Materials and Methods . These groups were MSCs alone (M), EPCs alone (E), MSCs +EPCs (M+E), BMP2 gene-modified MSCs (B2/M), BMP2 gene-modified EPCs (B2/E), EPC+BMP2 gene-modified MSCs (B2/M+E), MSC+BMP2 gene-modified EPCs (B2/E+M), and BMP2 gene-modified MSCs+BMP2 gene-modified EPCs (B2/(M+E)). The cells were induced with EGM/OS media for 14 days for qRT-PCR analysis. Data represent the mean+SD of n = 6 samples. (A) Osteoblast marker genes: OCN, Col I and BMP2. (B) Endothelial cell marker genes: VEGF, cdh5 and vWF. For A and B, ND: p>0.05, there is no significant difference between any two groups among the indicated ND groups. * p<0.05: between any two groups except the ND groups. # p<0.05: any ND groups vs. any other groups. ▾ p<0.05, any group in ND groups vs. any other groups.

Figure 4. Bone mineral density analysis for bone growth in rat critical-sized calvarial defect model.

(A) Images of calvarial bone obtained using LUNAR PIXImus system, 5 weeks after surgery. Upper row: general x-ray view. Lower row: x-ray with bone regeneration areas outlined in different colors. The black area circled with green lines in the implanted region is the low density area (fibrous tissue). The dark gray area between the green line and the yellow lines in the implanted region is the bone area with BMD that is lower than normal BMD. The area between the two yellow lines is considered the normal BMD area; bone density in this area is close to the BMD of normal bone tissue. (B) Quantitative analysis of bone density from A. The average BMD of normal intact bone is 0.0245 g/cm². *p<0.05: nCS/A+B2/(M+E) vs. each of the other five groups.

Figure 5. Bone growth in rat critical-sized calvarial defect model.

(A-E and A’-E’) Hematoxylin and eosin staining of calvarial bone for analysis of the new bone formation. Coronal sections through the midline of defects are shown. Margins of the original 8.0 mm trephine defect are shown. (A-E) lower magnification, bar = 1 mm. (A’-E’) higher magnification, bar = 0.5 mm. (A, A’): nCS/A; (B, B’): nCS/A+M; (C, C’): nCS/A +M+E; (D, D’): nCS/A+B2/M; (E, E’): nCS/A+B2/(M+E). Black arrow: newly formed bone. Blue arrow: blood vessels. (F) Quantitative analysis of bone area in implanted region from (A-E). BV, bone area in the implant; TV, total implant area. *p<0.05: nCS/A+B2/(M+E) vs. each of the other four groups. #p<0.05; nCS/A+M+E vs. nCS/A+B2/M or nCS/A+M or nCS; ▵p<0.05: nCS/A+B2/M vs. nCS/A+M or nCS/A only; ▿p<0.05: nCS/A+M vs. nCS/A.

Figure 6. Blood vessel growth in rat critical-sized calvarial defect model.

(A-E) Immunostaining of endothelial cell marker - vWF. (A’-E’) Immunostaining of VEGF. (A, A’): nCS/A; (B, B’): nCS/A+M; (C, C’): nCS/A+M+E; (D, D’): nCS/A+B2/M; (E, E’): nCS/A+B2/(M+E). (F) Quantitative analysis of blood vessel density from (A-E). (G) Quantitative analysis of blood vessel density from (A’-E’). N = 5, bar = 0.25 mm. *p<0.05: nCS/A+B2/(M+E) vs. each of the other four groups. #p<0.05: nCS/A+M+E vs. nCS/A+B2/M or nCS/A+M or nCS; ▵p<0.05: nCS/A+B2/M vs. nCS/A+M or nCS/A only; ▿p<0.05: nCS/A+M vs. nCS/A.